Objective: Develop and adapt a differentiation paradigm to produce enriched hiPSC-derived midbrain dopaminergic neurons (mDA), to resolve the temporal sequence of pathophysiological events in synucleinopathies.

Background: Mutations in the SNCA gene cause autosomal dominant Parkinson’s disease (PD), with progressive loss of dopaminergic neurons, and accumulation of aggregates of α-synuclein. However, the sequence of molecular events that proceed from the SNCA mutation to its end stage pathology is unknown. An enriched neuronal model to study the emergence of pathophysiological events is critical in understanding the causative events in synucleinopathies.

Method: We characterised our model using single-cell RNA-sequencing. We elucidated pathology in patient-derived mDA neurons using advanced single molecule super-resolution microscopy (DNA-PAINT, dSTORM) and a range of single cell live-cell imaging techniques.

Results: Our paradigm generates highly enriched midbrain dopaminergic (mDA) neurons (>80%), which are molecularly characterised using single-cell RNA sequencing. Functionally, neurons synthesize dopamine and generate action potentials. RNA-velocity analyses confirmed the developmental transcriptomic trajectory of neural precursors into mDA neurons using our approach; identifying key driver genes in mDA neuron development. At one week of differentiation, prior to molecular and functional identity, we demonstrate the formation of small aggregates; specifically, β-sheet rich oligomeric aggregates, in SNCA-mutant mDA immature neurons. The aggregation progresses over time to accumulate phosphorylated aggregates, and later fibrillar aggregates. When the mDA neurons are functional, we observed evidence of impaired physiological calcium signalling, with raised basal calcium, and impairments in cytosolic and mitochondrial calcium efflux. SNCA-mutant neurons then exhibited bioenergetic impairments, mitochondrial dysfunction and oxidative stress. Ultimately these multiple cellular stresses lead to an increase in cell death by six weeks post-differentiation.

Conclusion: Our differentiation paradigm generates an efficient model for studying disease mechanisms in PD, and highlights that protein misfolding to generate intraneuronal oligomers is one of the earliest critical events driving disease in human neurons, rather than a late-stage hallmark of the disease.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/protein-aggregation-and-calcium-dysregulation-are-the-earliest-hallmarks-of-synucleinopathy-in-enriched-ipsc-derived-human-midbrain-dopaminergic-neurons/